Synthesis, and evaluation of photophysical properties of a potential DPP-derived photosensitizer for photodynamic therapy with D-A-D architecture

The study of a macromolecule derived from DPP and triphenylamine, (DPP-BisTPA) by computational chemistry, its synthesis by direct arylation, optical characterization (UV-Vis and fluorescence) and electrochemistry (cyclic voltammetry), as well as its evaluation as a generator of reactive oxygen species indirectly, through the degradation of uric acid. The results obtained by DFT using B3LYP/6-31G (d, p) and TD-DFT using CAM-B3LYP/6-31G (d, p) reveal values of energy levels of the first singlet and triplet excited state that indicate a possible intersystem crossover and the possible generation of reactive oxygen species by a type I mechanism. The compound presents an absorption region within the phototherapeutic window. The electrochemical bandgap is 1.64 eV which suggests a behavior as a semiconductor. DPP-BisTPa were processed as hemispherical nanoparticles with a size around 100 nm, and NPOs were evaluated as a photosensitizer with a ROS generation yield of 4% using a photodynamic therapy flashlight as the light source. Graphical Abstract

Photodynamic therapy is a minimally invasive technique that has gained great popularity in recent years in treating cancer [15][16][17], mainly those that can be irradiated with light or use a light probe to carry out their processes [4,15,16].This consists of internalizing nanomaterials called photosensitizers that are activated by light, promoting an excited state that leads to intersystem crossing (ISC) and subsequent relaxation from a triplet state that interacts with cellular molecular oxygen to generate reactive oxygen species (ROS).such as singlet oxygen ( 1 O 2 ), hydroxyl radicals, the superoxide anion and hydrogen peroxide and thereby promote cell apoptosis [4,15].In this context, this is seeking to obtain photosensitizers with an excellent intersystem crossing (ISC) rate, minimal cytotoxicity in the dark, high molar extinction coefficient, minimally between 400 and 600 nm and that absorb in the so-called phototherapeutic region that is between 600 and 800 nm [16,17].
This article reports the synthesis, optical and electrochemical characterization and evaluation of the ability of a DPP-derived macromolecule with quadrupolar D-A-D architecture to generate reactive oxygen species as a potential photosensitizer material for photodynamic therapy.

Molecular design and theoretical calculations
Density functional theory (DFT) calculations have been performed using Gaussian 09w [66,67].To obtain the optimized structure and the frontier orbitals, B3LYP/ 6-31G (d, p) level of theory was used; To obtain the linear transitions of absorption and mission from the optimized molecular structures, TDDFT was used at the CAM-B3LYP/ 6-31 + G (d, p) level of theory with CPCM solvent model (chloroform).

Characterization
NMR spectra were performed on a Varian 300 mHz spectrometer.Infrared spectra were performed with ATR using a Perkin Elmer spectrophotometer.Ultraviolet absorption (UV) spectra (in chloroform solution and film) were recorded using a Perkin Elmer Lambda XLS spectrometer Perkin Elmer Lambda XLS.The fluorescence spectra (in chloroform solution and film) were obtained using a Perkin-Elmer LS55 spectrophotometer.Cyclic voltammetry (CV) measurement was performed on a Princeton Applied Research Potentiostat/Galvanostat Model 263 A electrochemical instrument with a 3-electrode cell in a solution of 0.1 M tetrabutylammonium hexafluorophosphate (Bu 4 NPF 6 ) in anhydrous acetonitrile at room temperature under nitrogen atmosphere with a scan rate of 50 mV/s.The working electrode was ITO with deposited DPP-BisTPA film, platinum wire as the auxiliary electrode and as reference electrode as an Ag/AgCl electrode.Ferrocene-ferrocenium (Fc/ Fc + ) couple was chosen as the internal standard.For the determination of the electrochemical values of the ionization potential (Ip) and the electronic affinity (Ea) as reported in the literature [68].The zeta potential of the dilute suspensions was measured with a Malvern Panalytical.The micrographs were obtained by Scanning Electron Microscope Quanta FEG-250 SEM instrument.

Organic nanoparticle manufacturing
Aqueous suspensions were prepared by reprecipitation method: 1 mL of DPP-BisTPA dissolved in THF (1.13 × 10−4 M) was injected into an aqueous solution of cetyltrimethylammonium bromide (CTAB) (10 mL) at a concentration of 8 × 10−4 M under ultrasonic stirring for 30 min.The THF present was evaporated by bubbling in nitrogen and ultrasonic stirring for 30 min, the procedure was repeated three times.3 Results and discussion

Synthesis
DPP-BisTPA was obtained according to the scheme, in Fig. 1.The product was purified obtaining 84% yield of the reaction, the product was characterized by FT-IR and 1 H NMR spectroscopies.
Figure 2 shows the FTIR spectrum of DPP-BisTPA and its raw materials, in particular the appearance of the bands of the

DFT study
The values of the energy levels of the HOMO and LUMO frontier orbitals were determined from the optimized structure of DPP-BisTPA. Figure 4 shows the isoimage of the difference between the excited and ground states of DPP-BisTPA.It can be seen that the charge transfer processes take place mainly in the central part of the molecule, which includes the DPP segment, the rings of the thiophene and the first phenyl of the triphenylamine, where planarity is lost.
Figure 5 shows the absorption spectra obtained from the TD-DFT calculation and in Table 1 the most probable electronic transitions.Based on the estimate, DPP-BisTPA presents two main electronic transitions, the first around 360 nm attributed to a transition between HOMO-2 and LUMO; and the second at 530 nm attributed to a typical transition from HOMO to LUMO energy levels [30,69], resulting in a possible intramolecular charge transfer [70,71].Although the planarity is affected, there are charge transfer processes from the triphenylamine to the central receptor moiety of the DPP.
The energy gap between the first singlet and triplet excited state (Δ S1→T1 ) is important to be able to predict a possible intersystem crossing (ISC) that allows having a triplet excited state, a reduction in Δ S1→T1 favors ISC [72].In Table 2 the estimated parameters are deposited, the Δ S1→T1 indicate a possible intersystem crossing, given the value of the triplet state, which could be the result of ICT processes.
When the intersystem crossing is performed, the mechanism by which the ROS are produced can be estimated.There are two main mechanisms of the photodynamic reaction, either the excited triplet state of the photosensitizer can transfer electrons to the surrounding triplet state molecular oxygen to generate free radicals such as hydroperoxides, superoxides or hydroxyl radicals (type I), or it can transfer its energy to a ground state triplet oxygen to generate excited state singlet oxygen ( 1 O 2 ) (type II) when the transferred energy exceeds 0.98 eV [41,73].In each case, different types of cell death are promoted, depending on the intracellular position of the photosensitizer.Damage to mitochondria in the cell membrane and damage to lysosomes or the endoplasmic reticulum can lead to apoptosis, necrosis and autophagy, respectively [16].Based on what has been described previously, DPP-BisTPA could generate ROS by mechanism I, it would generate free radicals because the estimated energy of its triplet state does not reach 0.98 eV.

Optical characterization properties
In Fig. 6 the absorption and emission spectra of DPP-BisTPA in solution, film and suspension are presented, two main electronic transitions can be observed, the most important being the most red-shifted.This is consistent with that estimated by TD-DFT, indicating possible charge transfer processes, which influence the value of the optical bandgap, which was estimated on film with a value of 1.53 eV, which is an excellent value for a conjugated system, entered within the range of organic semiconductors of interest for advanced applications.The maximum absorption wavelength of DPP-BisTPA processed as NPOs in aqueous suspension is 657 nm within the phototherapeutic window.
Table 3 shows the photophysical properties of DPP-BisTPA, the bandgap is around 1.5 eV, which is a very good value related to the region where it absorbs, the low fluorescence quantum yield indicates that the main process of relaxation is vibrational non-radiative, the intersystem crossover process is a process favored by vibrational relaxation.The reduction of the fluorescence quantum yield concerning the moiety of DPP precursor is attributed to an efficient ISC process [72,[74][75][76].In this case, it may be due to the intramolecular rotation of the triphenylamine moieties.In the solid state (nanoparticles), the ISC process usually increases, which favors the generation of singlet oxygen, since a triplet state of the photosensitizer is required, which, when relaxed, stimulates cellular molecular oxygen [77].processes around 1 eV, and reduction processes starting around −1eV, the frontier molecular orbitals were estimated, being −5.28 and 3.55 eV for HOMO and LUMO respectively, with an electrochemical bandgap of 1.73 eV, this corroborates the behavior as a possible organic semiconductor of DPP-BisTPA, as can be seen in Table 4.

Morphological characterization
Figure 8a presents a micrograph of the particles obtained in the reprecipitation process, as can be seen, the morphology of the reprecipitated particles is hemispherical with sizes around 100 nm (Fig. 8b), this size would allow internalization within the cancer cell to carry out photodynamic therapy [15].Figure 8c shows the graph of potential Z, and Table 5 condenses the results of this study.Based on the results obtained, it can be affirmed that the DPP-BisTPA particles have good stability since the colloidal suspension presents a Z potential of 47.3 mV and it is reported that the nanoparticles with zeta potential values greater than ± 30 have high degrees of stability [78,79].Also, with electrophoretic mobility of 3.7 µmcm/Vs and conductivity of 0.149 ms/cm.

Uric acid test
Figure 9 shows the uric acid degradation process through the action of DPP-BisTPA, for which a system within an absorption cell was used, it can be observed how the transition around 290 nm decreases with the step of time when in contact with the ONPs of DPP-BisTPA and under the irradiation of the phototherapy flashligth.Figure 9b shows the degradation kinetics.Table 6 summarizes the first kinetic parameter of uric acid degradation.It is worth    mentioning that the stability of uric acid was tested under irradiation conditions in buffer without the ONPs to verify that there is no photodegradation without the presence of ONPs of DPP-BisTPA.Likewise, tests were carried out previously with a white LED and a 532 nm green laser with a 3 mm beam waist and a power of 0.71 mW without having the effect of generating ROS, this is found in the supplementary material.This causes DPP-BisTPA to be activated only in the region of the phototherapeutic window using a dedicated PDT light source.The ONPs of DPP-BisTPA in exhibit a singlet oxygen generation yield of 4%.Possibly the energy gap between the first singlet excited state and the triplet is not short enough to make this process more efficient, which if carried out more efficiently would increase the generation of ROS.Although photosensitizers with yields between 2 and 4% have been reported [65,80,81], an attractive feature of DPP-BisTPA is that this activity as a photosensitizer has only been performed upon irradiation with light used in photodynamic therapy, thus avoiding processes with sources with wavelengths in the other regions of the visible spectrum.

Conclusions
DPP-BisTPA, a molecule that exhibits good absorption properties, and bandgap, was synthesized.The ONPs obtained have hemispherical morphology with sizes around 100 nm, presenting absorption within the phototherapeutic window.DPP-BisTPA exhibits a ROS generation yield of 4%, this is possibly related to the absorption capacity of the molecule, since it is not so intense above 650 nm, since the relaxation processes are favored for it to be vibrational.unless the energy gap in the singlet and triplet excited states is somewhat distant.However, ROS generation takes place only when irradiated with a light source within the phototherapeutic window, which prevents the effects of photosensitivity to sunlight when treated with PDT.The ROS generation results are consistent with the computationally estimated.Therefore, DPP-BisTPA is a candidate to be evaluated as a photosensitizer in photodynamic therapy.

Fig. 2
Fig. 2 FTIR spectra of DPP-BisTPA and its raw materials

Figure 3
shows the NMR spectrum of DPP-BisTPA identifying the signals of the protons of the aliphatic chain bound to the nitrogen of the DPP ring and the signals of the protons of the aromatic rings, integrating the number of expected protons.

Fig. 4
Fig. 4 Isoimage of the difference density between the ground state and the excited state of DPP-BisTPA

Figure 7
Figure 7 shows the cyclic voltammogram of DPP-BisTPA, a quasi-reversible process is observed, with oxidation

Fig. 6 a
Fig. 6 a Normalized absorption of DPP-BisTPA in THF solution, deposited on film and aqueous suspension of its NPOs, b normalized emission spectrum of DPP-BisTPA in THF solution, deposited on film and aqueous suspension of its NPOs

Table 1
Electronic transition data obtained by the CAM-B3LY´P/6-31 G (d, p)

Table 4
Electrochemical properties of DPP-BisTPA

Table 5 Z
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